Live-cell imaging studies of central nervous system synapse formation and alteration.

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Organization and dynamic remodeling of postsynaptic density (PSD) are thought to be important for postsynaptic signal transduction. To study the molecular mechanisms, we performed live-cell imaging analyses of dynamic characteristics of major PSD proteins both at steady state and when neurons are activated by exogenous stimulations.Cortactin is an F-actin-associated protein which interacts with the postsynaptic scaffolding protein Shank and is localized within the dendritic spine. Green fluorescent protein (GFP)-based time-lapse imaging revealed cortactin redistribution from dendritic cytoplasm to postsynaptic sites by application of brain-derived neurotrophic factor (BDNF). In contrast, activation of N-methyl-d-aspartate (NMDA) receptors induced loss of cortactin from postsynaptic sites. These results indicate the importance of counterbalance between BDNF and NMDA receptor-mediated signaling in the reorganization of the postsynaptic actin cytoskeleton during neuronal development.We also reported that four major scaffolding molecules, PSD-95, GKAP, Shank, and PSD-Zip45, show distinct instability in total molecular content per synapse. Fluorescence recovery after photobleaching (FRAP) also confirmed their distinct turnover rates. Among the PSD molecules examined, PSD-95 was most stable. Acute pharmacological disruption of F-actin eliminated the dynamic fraction of GKAP, Shank, and PSD-Zip45. GKAP content in synapses increased after pharmacological enhancement of neuronal activity, on the other hand Shank and PSD-Zip45 content are reduced. Inhibition of F-actin dynamics prevented activity-dependent redistribution of all three scaffolds. These results indicate that F-actin plays a key role for regulating the dynamic reorganization molecular composition in PSD.